Piezo1 Protein Research Articles

Mechanosensitive Piezo1 ion channel protein (PIEZO1 gene): update and extended mutation analysis of hereditary xerocytosis in India.

Hereditary xerocytosis (HX), also known as dehydrated stomatocytosis (DHSt) is a dominantly inherited genetic disorder exhibiting red cell membrane dehydration caused by the loss of the monovalent cation K+ and water. Variants in mechanosensitive Piezo ionic channels of the PIEZO1 gene are the primary cause of HX. We have utilized high throughput and highly precise next-generation sequencing (NGS) to make a diagnosis and examine the genotype-phenotype relationship in inflexible HX cases. Seven unrelated patients with unexplained hemolytic anemia were scrutinized with a panel probing 8000 genes related to congenital anemia. Targeted next-generation sequencing identified 8 missense variants in the PIEZO1 gene in 7 unrelated Indian patients. Three of the 8 variants are novel (c.1795G > C, c.2915G > A, c.7372T > C) and the remaining five (c.4082A > G, c.6829C > A, c.7374C > G, c.7381G > A, c.7483_7488dup) are previously reported. The variants have been validated by Sanger sequencing. One patient with autosomal dominant mutation (c.7372T > C) is associated with iron refractory iron deficiency anemia. Of the 7 patients, one has HX in combination with a novel homozygous variant (c.994G > A) in the PKLR gene causing PK deficiency resulting in severe clinical manifestations with phenotypic variability. In silico prediction using bioinformatics tools were used to study the possible damaging effects of the novel variants. Structural-functional analysis of the novel variants was investigated by molecular modeling software (PyMOL and Swiss PDB). These results encompass the heterogeneous behavior of mechano-sensitive Piezo1 protein observed in HX patients in India. Moreover, NGS imparted a subtle, economical, and quick tool for understanding the genetic cause of undiagnosed cases of congenital hemolytic anemia. NGS grants a potential technology integrating clinical history together with molecular report profiting in such patients and their families.

Chemical activation of the Piezo1 channel drives mesenchymal stem cell migration via inducing ATP release and activation of P2 receptor purinergic signaling.

In this study, we examined the Ca2+‐permeable Piezo1 channel, a newly identified mechanosensing ion channel, in human dental pulp‐derived mesenchymal stem cells (MSCs) and hypothesized that activation of the Piezo1 channel regulates MSC migration via inducing ATP release and activation of the P2 receptor purinergic signaling. The Piezo1 mRNA and protein were readily detected in hDP‐MSCs from multiple donors and, consistently, brief exposure to Yoda1, the Piezo1 channel‐specific activator, elevated intracellular Ca2+ concentration. Yoda1‐induced Ca2+ response was inhibited by ruthenium red or GsMTx4, two Piezo1 channel inhibitors, and also by Piezo1‐specific siRNA. Brief exposure to Yoda1 also induced ATP release. Persistent exposure to Yoda1 stimulated MSC migration, which was suppressed by Piezo1‐specific siRNA, and also prevented by apyrase, an ATP scavenger, or PPADS, a P2 generic antagonist. Furthermore, stimulation of MSC migration induced by Yoda1 as well as ATP was suppressed by PF431396, a PYK2 kinase inhibitor, or U0126, an inhibitor of the mitogen‐activated protein kinase MEK/ERK signaling pathway. Collectively, these results suggest that activation of the Piezo1 channel stimulates MSC migration via inducing ATP release and subsequent activation of the P2 receptor purinergic signaling and downstream PYK2 and MEK/ERK signaling pathways, thus revealing novel insights into the molecular and signaling mechanisms regulating MSC migration. Such findings provide useful information for evolving a full understanding of MSC migration and homing and developing strategies to improve MSC‐based translational applications.

Effect of knocking down Piezo1 mechanically sensitive protein on migration of MC3T3-E1 osteoblast cells

To discuss the effect of Piezo1 mechanically sensitive protein in migration process of mouse MC3T3-E1 osteoblast cells. The 5th-10th generation mouse MC3T3-E1 osteoblasts were divided into Piezo1-small interfering RNA (siRNA) transfection group (group A), negative control group (group B), and blank control group (group C). Piezo1-siRNA or negative control siRNA was transfected into mouse MC3T3-E1 osteoblasts by siRNA transfection reagent, respectively; group C was only added with siRNA transfection reagent; and the cell morphology was observed under inverted phase contrast microscope and fluorescence microscope, and the transfection efficiency was calculated. The expression of Piezo1 protein was detected by immunofluorescence staining and Western blot. Transwell cell migration assay and cell scratch assay were used to detect the migration of MC3T3-E1 osteoblasts after Piezo1-siRNA transfection. After 48 hours of transfection, group A showed a slight increase in cell volume and mutant growth, but cell colonies decreased, suspension cells increased and cell fragments increased when compared with untransfected cells. Under fluorescence microscope, green fluorescence was observed in MC3T3-E1 osteoblasts of group B, and the transfection efficiency was 68.56%±4.12%. Immunofluorescence staining and Western blot results showed that the expression level of Piezo1 protein in group A was significantly lower than that in groups B and C ( P<0.05); there was no significant difference between group B and group C ( P>0.05). Transwell cell migration assay and cell scratch assay showed that the number of cells per hole and the scratch healing rate of cells cultured for 1-4 days in group A were significantly lower than those in groups B and C ( P<0.05); there was no significant difference between group B and group C ( P>0.05). Piezo1 knocked down by siRNA can inhibit the migration ability of MC3T3-E1 osteoblast cells.

Retracted] Piezo1 protein induces the apoptosis of human osteoarthritis‑derived chondrocytes by activating caspase‑12, the signaling marker of ER stress.

The authors have requested that their research article entitled 'Piezo1 protein induces the apoptosis of human osteoarthritis‑derived chondrocytes by activating caspase‑12, the signaling marker of ER stress' published in International Journal of Molecular Medicine40, 845‑853, 2017, be retracted from the journal. Following the publication of this article, an interested reader drew to our attention that certain of the figures, or figure parts, published in this article contained data that also appeared at around the same time in a publication featuring several of the same authors in Experimental Cell Research: LiXF, Leng P, ZhangZ and ZhangHN: 'The Piezo1 protein ion channel functions in human nucleus pulposus cell apoptosis by regulating mitochondrial dysfunction and the endoplasmic reticulum stress signal pathway'. Exp Cell Res358: 377‑389, 2017. Though these studies were performed in osteoarthritic chondrocytes and nucleus pulposus cells, respectively, the authors realize that their submission to International Journal of Molecular Medicine was in breach of the requirement that a submitted paper should represent an (entirely) original study presenting novel work, and that none of the data therein had been previously submitted to or accepted by any other journal. Therefore, following the advice of the Editor, they are going to retract the paper from International Journal of Molecular Medicine. All the named authors agree to this retraction. They sincerely apologize for this mistake, thank the reader of their article who drew this matter to their attention, and regret any inconvenience this has caused. [the original article was published in International Journal of Molecular Medicine Med 40: 845-853, 2017; DOI: 10.3892/ijmm.2017.3075].

Expression characteristics of Piezo1 protein in stress models of human degenerative chondrocytes

To explore the expression characteristics of new mechanosensitive ion channel Piezo1 protein in stress models of human degenerative chondrocytes. The stress stimulation model of human degenerative chondrocytes in vitro was constructed. Multi-channel cell stretch stress loading system FX-4000T was used to treat chondrocytes. According to the results of pre-test, the loading frequency of 0.5 Hz and the cell elongation of 20% were loaded. According to cell processing time, it was divided into 0 h, 2 h, 12 h, 24 h and 48 h mechanical stress group. The RT-PCR and Western-blot were used to test the expression of the Piezo1, also the Laser scanning confocal microscope (LSCM) was used to test the intensity of the fluorescence of the Piezo1. (1)The result of the RT-PCR showed that the expression of the Piezo1 in the 2 h group was higher than the 0 h group(F=13.917, q=0.037 1, P<0.05). The expression of the piezo1 in the 24 h group was the highest. While the expression of the piezo1 in the 48 h group was lower than the expression of the piezo1 in the 24 h group(F=13.917, q=0.049 5, P<0.05). (2)The result of the Western-blot showed that the 2 h group was higher than the 0 h group(F=19.341, q=0.037 1, P<0.05). The expression of the 24 h had the highest expression which was higher than the 48 h group(F=19.341, q=0.017 7, P<0.05). (3)The Piezo1 protein was extensively expressed in the cytoplasm and nucleus of the nucleus pulposus cells. And with the increase of stress processing time, the fluorescence intensity of the protein also increased. In human degeneration cartilage cells, the new mechanio sensitive ion channel Piezo1 protein has a trace expression. After loading periodic mechanical tensile force, the expression of Piezo1 protein increases with time dependence.

Mechanosensitive ion channel Piezo1 is expressed in antral G cells of murine stomach

G cells in the antrum region of the murine stomach produce gastrin, the central hormone for controlling gastric activities. Secretion of gastrin is induced mainly by protein breakdown products but also by distensions of the stomach wall. Although G cells respond to protein fragments via distinct chemosensory receptor types, the mechanism underlying G cell activation upon distention is entirely ambiguous. Mechanosensitive ion channels are considered as potential candidates for such a task. Therefore, we explore the possibility of whether Piezo1, a polymodal sensor for diverse mechanical forces, is expressed in antral G cells. The experimental analyses revealed that the vast majority of G cells indeed expressed Piezo1. Within flask-like G cells at the base of the antral invaginations, the Piezo1 protein was primarily located at the basolateral portion, which is thought to be the release site for the exocytic secretion of gastrin. In the spindle-like G cells, which are oriented parallel to the invaginations, Piezo1 protein was restricted to the cell body where the hormone was also located, whereas the long processes appeared to be devoid of Piezo1 protein. Our results suggest that mechanosensitive channels such as Piezo1, located in close proximity to hormone-release sites, enable G cells to respond directly to antrum distensions with gastrin secretion.

The Function of the Novel Mechanical Activated Ion Channel Piezo1 in the Human Osteosarcoma Cells

BackgroundThe Piezo1 protein ion channel is a novel mechanical activated ion channel which is related to mechanical signal transduction. However, the function of the mechanically activated ion channel Piezo1 had not been explored. In this study, we explored the function of the Piezo1 ion channel in human osteosarcoma (OS) cells related to apoptosis, invasion, and the cell proliferation.Material/MethodsReverse transcription polymerase chain reaction (RT-PCR) and western-blotting were used to detect the expression of the Piezo1 protein. CCK-8, Transwell experiments and AV-PI were used to detected cell proliferation, cell invasion and cell apoptosis.ResultsThe Piezo1 protein ion channel was highly expressed in human OS cells. The Piezo1-shRNA inhibited the expression of the Piezo1. We explored whether LV3-PIEZO1-homo-3201 could act as Piezo1-shRNA, which could then be an inhibitor of Piezo1. The expression of Piezo1 in the 2-hour stretch group were slightly higher than the 0-hour stretch group, and the difference was not statistically significant (n=3, p>0.05, one-way ANOVA). The apoptotic gene such as the Bax, BAD, caspase-3, and caspase-9 had the same characteristics as the Piezo1 expression under the stretch force. We also explored the invasion of Piezo1 in vivo using nude mice, and found that Piezo1-shRNA could inhibit the invasion of the OS cells.ConclusionsThe Piezo1 protein may be a novel, potential therapeutic target for OS.

Piezo1 channels sense whole body physical activity to reset cardiovascular homeostasis and enhance performance

Mammalian biology adapts to physical activity but the molecular mechanisms sensing the activity remain enigmatic. Recent studies have revealed how Piezo1 protein senses mechanical force to enable vascular development. Here, we address Piezo1 in adult endothelium, the major control site in physical activity. Mice without endothelial Piezo1 lack obvious phenotype but close inspection reveals a specific effect on endothelium-dependent relaxation in mesenteric resistance artery. Strikingly, the Piezo1 is required for elevated blood pressure during whole body physical activity but not blood pressure during inactivity. Piezo1 is responsible for flow-sensitive non-inactivating non-selective cationic channels which depolarize the membrane potential. As fluid flow increases, depolarization increases to activate voltage-gated Ca2+ channels in the adjacent vascular smooth muscle cells, causing vasoconstriction. Physical performance is compromised in mice which lack endothelial Piezo1 and there is weight loss after sustained activity. The data suggest that Piezo1 channels sense physical activity to advantageously reset vascular control.

Piezo1 protein induces the apoptosis of human osteoarthritis-derived chondrocytes by activating caspase-12, the signaling marker of ER stress.

The present study was carried out to determine whether the mechanically activated cation channel Piezo1 protein plays a role as a signaling pathway which causes the apoptosis of human chondrocytes. The chondrocytes were isolated, cultured, and then subjected to mechanical stretch force for 0, 2, 12, 24 and 48 h, respectively. The expression levels of Piezo1 and the apoptosis-related protein caspase-12 were assessed by reverse transcription-quantitative polymerase chain reaction, as well as the apoptosis-related genes, B cell lymphoma/leukemia-2 (Bcl-2), Bcl-associated X protein (Bax) and Bcl-2-associated death promoter (BAD). Lactate dehydrogenase (LDH) activity was used to discern dead cells. Piezo1 expression was determined by immunofluorescence. In addition, Piezo1 inhibitor, GsMTx4, was used to block the mechanically activated (MA) cation channel Piezo1, and served as a positive control. The results showed that the osteoarthritis (OA)-derived chondrocytes showed a tendency to undergo late-stage apoptosis under compressive loading. Piezo1 and caspase-12 were significantly upregulated under static compressive stimuli and the expression was related to the rate of apoptosis of the OA-derived chondrocytes during compressive loading. The expression of caspase-12 and late-stage apoptosis of the human OA-derived chondrocytes were repressed by GsMTx4, the specific inhibitor of Piezo1, while the expression of Piezo1 and the induction of the apoptosis of the OA-derived chondrocytes during compressive loading was not totally blocked. Thus, we conclude that Piezo1 plays an important role in the apoptosis of human OA-derived chondrocytes through a caspase-12-dependent pathway. The expression of Piezo1 protein was not totally inhibited by GsMTx4.

RETRACTED: The Piezo1 protein ion channel functions in human nucleus pulposus cell apoptosis by regulating mitochondrial dysfunction and the endoplasmic reticulum stress signal pathway

The Piezo1 protein ion channel is a novel mechanical stretch-activated ion channel (SAC) closely related to mechanical signals. Mechanotransduction plays a crucial role in organ development and homeostasis. Previous studies identified Piezo1 and demonstrated that it is distinct from other ion channels with well-established roles in lower organisms. Mechanical stretch-activated ion channels from other organisms are not conserved in mammals or do not act as mechanically activated channels in mammals. In the current study, we explored the role of the Piezo1 ion channel in human nucleus pulposus cell (NP cell) apoptosis through mechanical force-induced mitochondrial dysfunction and endoplasmic reticulum stress. Reverse Transcription Polymerase chain reaction (RT-PCR), immunofluorescence, immunohistochemistry and Annexin V binding and propidium iodide analyses revealed that the Piezo1 protein ion channel was highly expressed in human NP cells, which are the primary cells that comprise the intervertebral disc. In patients with intervertebral disc degeneration (IVDD), the Piezo1 protein may play a crucial role in human NP cell apoptosis through mitochondrial dysfunction and endoplasmic reticulum stress under abnormal loading conditions. This study also verified that human NP cells have an intimate connection with the cytoskeleton upon treatment of the cells with the Piezo1 blocking peptide GsMTx4 from tarantula venom. In summary, Piezo1 functions in human NP cell apoptosis, which may be one underlying mechanism of apoptosis induced by abnormal loading in IVDD patients.

Evidence for shear-mediated Ca2+ entry through mechanosensitive cation channels in human platelets and a megakaryocytic cell line

The role of mechanosensitive (MS) Ca2+-permeable ion channels in platelets is unclear, despite the importance of shear stress in platelet function and life-threatening thrombus formation. We therefore sought to investigate the expression and functional relevance of MS channels in human platelets. The effect of shear stress on Ca2+ entry in human platelets and Meg-01 megakaryocytic cells loaded with Fluo-3 was examined by confocal microscopy. Cells were attached to glass coverslips within flow chambers that allowed applications of physiological and pathological shear stress. Arterial shear (1002.6 s−1) induced a sustained increase in [Ca2+]i in Meg-01 cells and enhanced the frequency of repetitive Ca2+ transients by 80% in platelets. These Ca2+ increases were abrogated by the MS channel inhibitor Grammostola spatulata mechanotoxin 4 (GsMTx-4) or by chelation of extracellular Ca2+. Thrombus formation was studied on collagen-coated surfaces using DiOC6-stained platelets. In addition, [Ca2+]i and functional responses of washed platelet suspensions were studied with Fura-2 and light transmission aggregometry, respectively. Thrombus size was reduced 50% by GsMTx-4, independently of P2X1 receptors. In contrast, GsMTx-4 had no effect on collagen-induced aggregation or on Ca2+ influx via TRPC6 or Orai1 channels and caused only a minor inhibition of P2X1-dependent Ca2+ entry. The Piezo1 agonist, Yoda1, potentiated shear-dependent platelet Ca2+ transients by 170%. Piezo1 mRNA transcripts and protein were detected with quantitative RT-PCR and Western blotting, respectively, in both platelets and Meg-01 cells. We conclude that platelets and Meg-01 cells express the MS cation channel Piezo1, which may contribute to Ca2+ entry and thrombus formation under arterial shear.

The Structural Basis for Sensing bythe Piezo1 Protein.

Mechanotransduction is one of the processes by which cells sense and convert mechanical stimuli into biological signals. Experimental data from various species have revealed crucial roles for mechanotransduction in organ development and a plethora of physiological activities. Piezo proteins have recently been identified as the long-sought-after mechanically activated cation channels in eukaryotes. The architecture of mouse Piezo1 (mPiezo1) channel determined by cryoelectron microscopicsingle-particle analysis at medium resolution yielded important insights into the mechanical force sensing mechanism. mPiezo1 is found to form a trimeric propeller-like structure with the extracellular domains resembling three distal blades and a central cap. The transmembrane region consists of a central pore module that likely determines the ion-conducting properties of mPiezo1, and three peripheral wings formed by arrays of paired transmembrane helices. Compared with the central pore module, the three distal blades display considerably larger flexibility. In the intracellular region, three long beam-like domains (∼80Å in length) support the whole transmembrane region and connect the mobile peripheral regions to the central pore module. This unique design suggests that the trimeric mPiezo1 may mechanistically function in similar principles as how propellers sense and transduce force to control the ion conductivity. This review summarizes the current knowledge on the structure and proposes possible gating mechanisms of mPiezo1.

Mechanism of the Piezo1 protein-induced apoptosis of the chondrocytes through the MAPK/ERK1/2 signal pathway

To invstigate the mechanism of new mechanically-activated cation channel protein (Piezo1) can cause the apoptosis of the human chondrocytes under compressive loading, using a Flexercell unit by activating classical Mitogen-activated protein kinase (MAPK) signal pathyway(ERK1/2). Primary human chondrocytes were isolated, cultured, and then subjected to the static compressive loading for 0, 2, 12, 24, 48 h, respectively.The expressions of Piezo1 and the ERK1/2 were assessed by reverse transcription-polymerase chain reaction(PT-PCR), as well as the apoptosis gene B cell lymphoma/leukemia-2(Bcl-2) Bel-associated X protein(Bax). In addition, Piezo1inhibitor, Grammostola spatulata mechanotoxin 4(GsMTx4), was used to block Piezo1, served as a positive control.The immunofluorescence was used to locate the expression of Piezo1 protein and ERK1/2.AnnexinV-PI was used to detect the apoptosis of chondrocytes. The expression of the Piezo1 in chondrocytesis was weak, the 12 h group was significant increased(0.198 1 vs 0.021 4, P<0.05), the 24 h group was the highest expression while the expression of the 48 h group was lower than the 24 h group, as well as the ERK1/2, Bcl-2 and caspase3.The result of AV-PI had shown that the 2 h group had increased early stage of apoptosis.The 12 h group had increased late stage of apoptosis, and the 24 h group's apoptotic rate was the highest, while the apoptotic rate of the 48 h group was lower than the 24 h group(0.497 1 vs 0.743 1, q=0.035 9). The GsMTx4 could inhibit the late stage of apoptosis, and the location of the Piezo1 was expressed in the nucleus and cytoplasm of the chondrocytes. Piezo1 plays an important role in the apoptosis of the human chondrocyte through the classic MAPK/ERK1/2 signal pathway.

Mechanism of Piezo1 protein induced apoptosis of osteoarthritis chondrocytes through MAPK/ERK5 signal pathway

Objective To investigate whether the new mechanically-activated (MA) cation channel Piezo1 protein can cause the apoptosis of human chondrocytes under compressive loading, using a Flexercell unit by activating MAPK/ERK5 signal pathway. Methods Primary human chondrocytes were isolated, cultured, and then subjected to the static compressive loading for 2 h,12 h, 24 h and 48 h, respectively. The GsMTx4, which is the special inhibitor of the Piezo1 protein and the BIX02188, which is the inhibitor of the ERK5 served as the inhibitor group. The immunofluorescence was used to locate the expression of the Piezo1 protein. The expressions of Piezo1 and ERK5 were assessed by reverse transcription-polymerase chain reaction (RT-PCR), as well as the apoptosis gene B cell lymphoma/leukemia-2 (Bcl-2), Bclassociated X protein (Bax) and Bclassociated death promoter (BAD). In addition, Piezo1 inhibitor, GsMTx4, was used to block the MA cation channel Piezo1, served as the inhibitor group. AV-PI was used to detect the apoptosis of the OA chondrocytes. Results The location of the Piezo1 was expressed in nucleus and cytoplasm of chondrocytes. The expression of the Piezo1, ERK5, BAD, and Bax mRNA in the OA chondrocytes is weak. The 12 h stretch force group was significant increased, and the 24 h stretch force group was the highest expression. However, the expression of the 48 h group was decreasing. The expression of the Bcl-2 in the 12 h group was decreasing, and the 12 h stretch force group was the lowest expression while the 24 h stretch force group was increasing. In the GsMTx4 group, the expression of the Piezo1, ERK5, BAD, Bax was decreasing while the Bcl-2 was increasing. In the BIX02188 group, the expression of the ERK5, BAD, Bax was increasing while the Bcl-2 was decreasing, while the expression of the Piezo1 was not change. The result of AV-PI shown that the 2 h stretch force group increased early stage of apoptosis. The 12 h stretch force group increased late stage of apoptosis, and the 24 h stretch force group's apoptotic rate was the highest. However, the apoptotic rate of the 48 h group was lower than the 24 h stretch force group. The GsMTx4 could inhibit the late stage of apoptosis. Conclusion Piezo1 plays an important role in the apoptosis of human chondrocyte through the MAPK/ERK5 signal pathway. Key words: Ion channels; Chondrocytes; Osteoarthritis; MAP kinase signaling system; Apoptosis

Abstract 11593: Thrombus Formation Under Flow is Inhibited by the Mechanosensitive Cation Channel Blockers GsMTx-4 Peptide and Gadolinium Chloride

Introduction: Ion channels contribute significantly to platelet physiology, including control of Ca 2+ as a second messenger. Although there is a clear link between platelet activation and shear stress, no study has examined the functional relevance of mechanosensitive (MS) ion channels in platelets or their precursors, megakaryocytes. Hypothesis: Human platelets express MS cation channels such as Piezo1, which contribute to platelet function and the development of thrombosis. Methods: Thrombus formation and [Ca 2+ ] i (intracellular Ca 2+ ) responses were studied by confocal microscopy using the indicators DiOC 6 and Fluo-3, respectively, under physiological levels of shear stress in a parallel-plate flow chamber. Aggregation and [Ca 2+ ] i responses to collagen and thrombin in washed human platelets were assessed by light transmission aggregometry and Fura-2 ratiometric measurements. GsMTx-4 (2.5μM) and gadolinium chloride (GdCl 3 ) (30μM) were used as MS cation channel inhibitors, targeting Piezo1 and TRPC6 in platelets. Piezo1 mRNA and protein expression were studied in platelets and the megakaryocytic cell line Meg-01. Results: Both inhibitors significantly inhibited thrombus formation on a collagen surface under normal arterial shear rate (average thrombus volume was reduced from 48132.7±7739.4μm 3 to 13478.9±2744.8μm 3 by GdCl 3 (n=6, P&lt;0.01) and to 25125.9±6433.5μm 3 (n=6, P&lt;0.05) by GsMTx-4). GsMTx-4 also caused a reduction in thrombin-induced platelet aggregation (to 53.6±14.0% of control, n=4, P&lt;0.05), however GdCl 3 did not affect platelet shape change or aggregation under static conditions. Pre-treatment with either GsMTx-4 or GdCl 3 did not reduce calcium influx in response to TRPC6 stimulation with 60μM OAG, suggesting a MS role for Piezo1, which was found at the mRNA transcript and protein level in platelets and Meg-01 cells. Meg-01 cells showed shear rate-dependent increases in [Ca 2+ ] i levels; at a shear rate of 3989.2s -1 , F/F 0 increased to 1.38±0.05 from 1.00±0.00 baseline (n=53 cells, P&lt;0.0001), a response which was diminished by GsMTx-4, and by chelation of extracellular Ca 2+ by EGTA. Conclusions: We provide evidence for the first time for a functional contribution of MS cation channels in platelet physiology and thrombosis.

Channelizing the red blood cell: molecular biology competes with patch-clamp.

Channelizing the red blood cell: molecular biology competes with patch-clamp.

Piezo1 forms mechanosensitive ion channels in the human MCF-7 breast cancer cell line.

Mechanical interaction between cells – specifically distortion of tensional homeostasis-emerged as an important aspect of breast cancer genesis and progression. We investigated the biophysical characteristics of mechanosensitive ion channels (MSCs) in the malignant MCF-7 breast cancer cell line. MSCs turned out to be the most abundant ion channel species and could be activated by negative pressure at the outer side of the cell membrane in a saturable manner. Assessing single channel conductance (GΛ) for different monovalent cations revealed an increase in the succession: Li+ < Na+ < K+ ≈Rb+ ≈ Cs+. Divalent cations permeated also with the order: Ca2+ < Ba2+. Comparison of biophysical properties enabled us to identify MSCs in MCF-7 as ion channels formed by the Piezo1 protein. Using patch clamp technique no functional MSCs were observed in the benign MCF-10A mammary epithelial cell line. Blocking of MSCs by GsMTx-4 resulted in decreased motility of MCF-7, but not of MCF-10A cells, underscoring a possible role of Piezo1 in invasion and metastatic propagation. The role of Piezo1 in biology and progression of breast cancer is further substantiated by markedly reduced overall survival in patients with increased Piezo1 mRNA levels in the primary tumor.

Piezo1 is as a novel trefoil factor family 1 binding protein that promotes gastric cancer cell mobility in vitro.

Trefoil factor family 1 (TFF1) is a member of the TFF-domain peptide family involved in epithelial restitution and cell motility. Recently, we screened Piezo1 as a candidate TFF1-binding protein. We aimed to confirm Piezo1 as a novel TFF1 binding protein and to assess the role of this interaction in mediating gastric cancer cell mobility. This interaction was confirmed by co-immunoprecipitation and co-localisation of TFF1 and Piezo1 in GES-1 cells. We used stable RNA interference to knockdown Piezo1 protein expression and restored the expression of TFF1 in the gastric cancer cell lines SGC-7901 and BGC-823. Cell motility was evaluated using invasion assay and migration assay in vitro. The expression levels of the integrin subunits β1, β5, α1 as well as the expression of β-catenin and E-cadherin were detected by Western blot. We demonstrate that TFF1, but not TFF2 or TFF3, bind to and co-localize with Piezo1 in the cytoplasm in vitro. TFF1 interacts with the C-terminal portion of the Piezo1 protein. Wound healing and trans-well assays demonstrated that the restored expression of TFF1 promoted cell mobility in gastric cancer cells, and this effect was attenuated by the knockdown of Piezo1. Western blots demonstrated the decreased expression of integrin β1 in Piezo1-knockdown cells. Our data demonstrate that Piezo1 is a novel TFF1 binding protein that is important for TFF1-mediated cell migration and suggest that this interaction may be a therapeutic target in the invasion and metastasis of gastric cancer.

Two Segments of the Human PIEZO1 Mechanosensitive Ion Channel Can Reassemble into a Functional Unit

We created clones that covalently linked a fluorescent protein to PIEZO1 in order to image PIEZO1 on the cell surface. Inserting mCherry1 in internal positions of the PIEZO1 protein, we found a construct at position 1591 that yielded a protein whose channel kinetics were nearly identical to wild type. Through cotransfection, we imaged PIEZO1 (mCherry) and TREK-1 (GFP) on HEK cells. These two mechanosensitive channels were at the cell surface in completely different physical domains. PIEZO1 appeared as random clusters but TREK tended to follow cytoskeleton tracks.Given the stability of the PEIZO1 protein with a fluorescent group at 1591, we asked if the PIEZO1 protein could be split at position 1591 and still function. Fragment 1 (1-1591) was fused to mCherry, and fragment 2 (1592-2520) to GFP. Co-expression of the two segments showed currents in whole cell recording, cell-attached patches, and outside-out patches. The biophysical characteristics were nearly identical to the wild type channel, having a reversal potential around 0 mV and voltage dependent inactivation in whole cell and cell attached mode.We next asked if either fragment alone was functional. The C-terminal fragment produced mechanosensitive currents in outside-out patches but the N terminal segment (1-1591) did not in any recording mode. We then shortened the C terminal fragment from its C- and N- termini using the outside-out patches to screen for current. We obtained mechanosensitive currents from a fragment of ∼ 450 amino acids, so the fundamental properties of the giant PIEZO1 channel are contained in this small fragment. This protein segment appears to contain the mechanosensor and the gate, and establishes that eight putative transmembrane domains are required for pore formation. None of the shortened fragments displayed whole cell currents.

Mechano-Sensitive Ion Channels (MSCS) Provide Human Breast Cancer Cells with a Sensorium for Mechanical Stress

Mechanical stress is increasingly recognized as a cancerogenic factor in breast cancer. We investigated the biophysical characteristics of mechano-sensitive channels (MSCs) in the breast cancer cell line MCF7 with patch clamp methods.MSCs were present in 132 out of 258 cell-attached membrane patches. MSCs could be activated by negative pressure at the outer side of the membrane in a saturable manner (EP50: 41.2 ± 0.5 mbar (N=13).When K+ was predominantly present in the extracellular pipette solution, single channel conductance exerted to be 25.6± 0.4 pS (N=8). When complete ion selectivity was assessed, conductivity was found to increase in the following order: Li+ < Na+ < K+≈Rb+ ≈ Cs+. Furthermore, conductivity was smaller for divalent cations (100 mmole/L compared to 153 mmole/L) with the order: Ca2+ < Ba2+ < K + ≈ Rb + ≈ Cs +. We compared the biophysical properties of MSCs with a recently discovered mechanosensitive cation channel, the human Piezo1 protein, heterologously overexpressed in HEK293 cells. Single channel conductances were indistinguishable between MSCs from MCF7 and hPiezo1 in HEK293 cells, both when 100% K + and 50% K + / 50% Na + were used as permeant ions.MSCs occur in MCF7 breasts cancer cells, providing a sensorium for mechanical stress. Ion selectivity studies show that divalents like Ca2+ can permeate to a considerable extend. Moreover, permeation of monovalents is apparently restricted by the size of the ions hydrate shell, as indicated by the smaller conductivities for Na + and Li +, respectively, indicating that the ion permeation mechanism through MSCs may be different to the cation channels crystallized so far.This research is supported by grants from the Austrian Research Foundation (FWF22974 (WS); KLIF182 (TB)).